A three-dimensional fluorescence spectrum obtained by a Fluorescence Spectrophotometer is effective to recognize an excitation wavelength, a fluorescence wavelength, and a fluorescence intensity of a fluorescence substance contained in a sample. However, when a fluorescence peak appears in the three-dimensional fluorescence spectrum, in a case where the fluorescence substance contained in the sample is unknown, it is not easy to specify what the substance is. Most of them are estimated from the peak information of the excitation wavelength and the fluorescence wavelength by referring to past report examples, but usually a sample contains a plurality of components, and in a case where peaks of the components overlap and a plurality of candidate peaks for the excitation wavelength and the fluorescence wavelength are located adjacent to each other, there may be no past report example, which hinders specification of a substance.
Recognizing the mass information of a component greatly contributes to substance identification. In a case where there is a plurality of candidate components in the past report example, it is possible to narrow down the candidates based on the mass information. In Li, Wen-Tao, et al. “Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: a comparative study.” Environmental science & technology 48.5 (2014): 2603-2609 (which will be referred to as NPL 1, an abbreviation of Non-Patent Literature 1), a method using a size exclusion column of a high-performance liquid chromatograph and fluorescence wavelength information of a fluorescence detector to obtain mass information has been proposed. Since an elution time changes for each molecular weight in the size exclusion column, it is possible to estimate the molecular weight, based on the elution time of a target sample, by measuring the calibration sample having a plurality of known molecular weights and recognizing the relationship between the molecular weight and the elution time in advance.
A three-dimensional fluorescence spectrum (an elution time, a fluorescence wavelength, and a fluorescence intensity) of each sample component separated by the column is measured by using a fluorescence detector as a detector of a liquid chromatograph, and it is determined whether the measured fluorescence wavelength matches the fluorescence wavelength of the target component of the three-dimensional fluorescence spectrum obtained by the Fluorescence Spectrophotometer, that is, it is a target component, so it is possible to estimate the molecular weight based on the elution time of the component (JP-A-2009-180706). In order to calculate the concentration of a component to be measured (aflatoxin) contained in the sample, the three-dimensional fluorescence spectrum is measured by adding an aflatoxin solution having a different concentration to the sample, so the relationship between the excitation fluorescence wavelength and the concentration is obtained and a calibration model is generated (Japanese Patent No. 5856741).
When a fluorescence peak appears in the three-dimensional fluorescence spectrum, in a case where a fluorescence substance contained in the sample is unknown, it is not easy to specify the substance. There are methods of JP-A-2009-180706 and Japanese Patent No. 5856741 in which molecular weight is estimated from elution time by measuring a calibration sample in a measurement system using a size exclusion column and a fluorescence detector, but in these methods, it is necessary to recognize in advance the relationship between the molecular weight and the elution time in the calibration sample having a plurality of known molecular weights, and since the molecular weight obtained is a predicted value for the calibration sample, the accuracy of molecular weight is not high. Therefore, as described in NPL 1, components are predicted in advance, it is effective when recognizing the characteristics of the molecular weight distribution with components such as proteins and humus substances, but it is not suitable when recognizing the molecular weight with unknown component estimation. When using a size exclusion column, there is a problem that it is complicated because it is necessary to prepare a plurality of calibration samples corresponding to the molecular weights after estimating the molecular weights to some extent and to optimize the elution condition according thereto.